Efficient synthesis of novel dialkyl-3-cyanopropylphosphate derivatives and evaluation of their anticholinesterase activity

Based on the broad spectrum of biological activities associated with organophosphates, a novel type of this class of compounds was synthesized, bearing a nitrile group, from the sodium alkoxide-catalyzed reaction of dialkylphosphites with γ-ketonitriles at 80 °C under solvent-free conditions. A reaction mechanism involving a phospha-Brook type rearrangement is proposed. Eight title compounds were investigated for their in vitro inhibitory potency and selectivity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) using Ellman’s spectrophotometric method. The synthesized derivatives exhibited mostly a moderate activity against both cholinesterases. The IC₅₀ values for BChE were in a smaller concentration range (5.96–23.35 µM) compared to those for AChE inhibition (9.61–53.74 µM). The diethyl-3-cyano-1-p-tolylpropylphosphate which displayed the higher dual inhibitory potency towards both cholinesterases could be considered as a potential candidate for developing new drugs to treat Alzheimer’s disease.     

The effect of environmental parameters on growth,cholinesterase activity and glutathione S-transferase activity in the earthworm (Apporectodea caliginosa)

The effect of age and environmental parameters on growth, cholinesterase (ChE), and glutathione S-transferase (GST) activities were assessed in juvenile Apporectodea caliginosa earthworms. Earthworms were maintained in three types of soil- loam, sandy, and clay- at a range of moisture contents (15-30%), and temperatures (5-20 C). Earthworm age (1-3 months) had no significant affect on ChE activity. Growth rates were influenced by all environmental parameters tested and these effects were interrelated. Optimum conditions for growth appeared to be in loam or sandy soil with 25-30% moisture at 10-15 C. The GST activity was influenced by soil temperature and activity was significantly higher at 15 C than at 5 C and 10 C. Soil type also influenced GST activity and this influence was dependent on moisture content. In sandy soil GST activity was significantly lower at 30% moisture than at lower moisture contents, in loam soil GST activity was significantly higher at 15% moisture than at higher moisture contents, while in clay soil GST activity was not affected by moisture content. These results indicate that in field experiments when evaluating GST activity soil temperature and soil type need to be consistent between control and 'contaminated sites'. ChE activity was only affected by temperature, so this should be considered when comparing control and treatedareas.     

Synthesis and biological evaluation of 4-arylcoumarins as potential anti-Alzheimer’s disease agents

Alzheimer's disease (AD) is a progressive neurological degenerative disease that has complex pathogenesis. A variety of studies in humans indicate that several enzymes inhibitors can be useful in the treatment of AD, including acetylcholinesterase (AchE), butyrylcholinesterase (BuChE) and monoamine oxidase (MAO). Various substituted 4-arylcoumarin derivatives were synthesised, and their activity in vitro were investigated, including AChE/BuChE inhibitory activity, MAO inhibitory activity, and antioxidant activity. Most of the compounds were found to exhibit high inhibitory activity, and individual compounds have extremely excellent activities. Therefore 4-arylcoumarins provides an idea for drugs design for the development of therapeutic or preventive agents for AD.     

Choline Acetyltransferase and Cholinesterases in the Developing Xenopus Retina

To understand the developmental regulation of acetylcholine (ACh) synthesis in the Xenopus retina, the properties of choline acetyltransferase (CAT) and cholinesterase (ChE), as well as histochemical localization of ChE in the retina, were studied during development. CAT activity first became detectable in the developing eyecup at stages 35/36. This was followed by a rapid, 50-fold rise in specific activity between stages 35/36 and 44. Since this rapid rise coincided with an almost identical increase in total ACh synthesis in whole retinae found in previous studies, it is suggested that this increase was sufficient to account for the rapid increase in total ACh synthesis. Moreover, it also correlated with increased rates of synaptogenesis in both the inner and the outer plexiform layers. Total ChE was resolved into specific and nonspecific ChE by the use of tetraisopropylpyrophosphoramide. Total ChE activities first became detectable at stages 35/36. Specific ChE [acetylcholinesterase (AChE)] increased from 50% at stage 39 to 95% of total ChE activities at stage 66. Again, the most rapid increase in both total ChE and AChE activities occurred between stages 35/36 and 44. Histochemical studies showed that AChE was localized predominantly in the two plexiform layers, with the inner plexiform layer more heavily stained at all stages. Moreover, a stratified staining pattern, clearly discerned in the inner plexiform layer, also correlated with synaptogenesis during this early period of retinal development.     

Donepezil treatment restores the ability of estradiol to enhance cognitive performance in aged rats: Evidence for the cholinergic basis of the critical period hypothesis

Recent studies suggest that the ability of estradiol to enhance cognitive performance diminishes with age and/or time following loss of ovarian function. We hypothesize that this is due, in part, to a decrease in basal forebrain cholinergic function. This study tested whether donepezil, a cholinesterase inhibitor, could restore estradiol effects on cognitive performance in aged rats that had been ovariectomized as young adults. Rats were ovariectomized at 3 months of age, and then trained on a delayed matching to position (DMP) T-maze task, followed by a configural association (CA) operant condition task, beginning at 12-17 or 22-27 months of age. Three weeks prior to testing, rats started to receive either donepezil or vehicle. After one week, half of each group also began receiving estradiol. Acclimation and testing began seven days later and treatment continued throughout testing. Estradiol alone significantly enhanced DMP acquisition in middle-aged rats, but not in aged rats. Donepezil alone had no effect on DMP acquisition in either age group; however, donepezil treatment restored the ability of estradiol to enhance DMP acquisition in aged rats. This effect was due largely to a reduction in the predisposition to adopt a persistent turn strategy during acquisition. These same treatments did not affect acquisition of the CA task in middle-aged rats, but did have small but significant effects on response time in aged rats. The data are consistent with the idea that estrogen effects on cognitive performance are task specific, and that deficits in basal forebrain cholinergic function are responsible for the loss of estradiol effect on DMP acquisition in aged ovariectomized rats. In addition, the data suggest that enhancing cholinergic function pharmacologically can restore the ability of estradiol to enhance acquisition of the DMP task in very old rats following long periods of hormone deprivation. Whether donepezil has similar restorative effects on other estrogen-sensitive tasks needs to be explored.     

Pressure-induced molten globule state of cholinesterase

The denaturing effect of pressure on the structure of human butyrylcholinesterase was examined by gel electrophoresis under pressure and by 8-anilino-1-naphthalene sulfonate (ANS) binding. It was found that the fluorescence intensity of bound ANS is increased by pressure between 0.5 and 1.5 kbar and that the hydrodynamic volume of the enzyme swells when pressures around 1.5 kbar are applied. These findings indicate that pressure denaturation of butyrylcholinesterase is a multi-step process and that the observed transient pressure-denatured states have characteristics of molten globules.     

Design,synthesis and anti-Alzheimer’s activity of novel 1,2,3-triazole-chromenone carboxamide derivatives

Alzheimer’s disease (AD) is a well-known neurodegenerative disorder affecting millions of old people worldwide and the corresponding epidemiological data highlights the significance of the disease. As AD is a multifactorial illness, various single-target directed drugs that have reached clinical trials have failed. Therefore, various factors associated with outset of AD have been considered in targeted drug discovery and development. In this work, a wide range of 1,2,3-triazole-chromenone carboxamides were designed, synthesized, and evaluated for their cholinesterase inhibitory activity. Among them, N-(1-benzylpiperidin-4-yl)-7-((1-(3,4-dimethylbenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-oxo-2H-chromene-3-carboxamide (11b) showed the best acetylcholinesterase inhibitory activity (IC₅₀ = 1.80 µM), however, it was inactive toward butyrylcholinesterase. It should be noted that compound 11b was evaluated for its BACE1 inhibitory activity and calculated IC₅₀ = 21.13 µM confirmed desired inhibitory activity. Also, this compound revealed satisfactory neuroprotective effect against H₂O₂-induced cell death in PC12 neurons at 50 µM as well as metal chelating ability toward Fe²⁺, Cu²⁺, and Zn²⁺ ions.     

Serum cholinesterase assay using a reagent-free micro pH-stat

Enzyme activities in body fluids are often used as diagnostic markers for physiological conditions and diseases. Common enzyme assays use optical methods that often require the use of pseudosubstrates and associated dyes. Here we introduce a reagent-free micro pH-stat that can determine absolute enzyme activity without the need for exogenic reagents. This approach employs electrolysis for precise dosing of the requisite acid or base titrant to stat the pH of the sample. The micro pH-stat is based on the rotating sample system (RSS), a convection platform for microliter drops. Activities of serum cholinesterase in fetal bovine serum and human serum were analyzed with this approach. The performance of this system is comparable to that of standard techniques (r² = 0.99), yet it offers a broader range of detection. The reagent-free micro pH-stat has potential to be developed as a miniaturized device for point-of-care testing.     

Dual functional cholinesterase and PDE4D inhibitors for the treatment of Alzheimer’s disease: Design,synthesis and evaluation of tacrine-pyrazolo[3,4-b]pyridine hybrids

A series of tacrine-pyrazolo[3,4-b]pyridine hybrids were synthesised and evaluated as dual cholinesterase (ChE) and phosphodiesterase 4D (PDE4D) inhibitors for the treatment of Alzheimer’s disease (AD). Compound 10j, which is tacrine linked with pyrazolo[3,4-b]pyridine moiety by a six-carbon spacer, was the most potent acetylcholinesterase (AChE) with IC₅₀ value of 0.125 μM. Moreover, compound 10j provided a desired balance of AChE and butylcholinesterase (BuChE) and PDE4D inhibition activities, with IC₅₀ value of 0.449 and 0.271 μM, respectively. The above results indicated that this hybrid was a promising dual functional agent for the treatment of AD.     

The protective role of parsley extract against vincristine mutagenicity in Drosophila melanogaster

In this study, Drosophila melanogaster males were treated with parsley plant extract and the anticancer drug vincristine (VCR) singly and in combined treatments (pre, co and post-treatments) to detect the mutagenic effects by using sex-linked recessive lethal test (SLRL) and estimation of cholinesterase enzyme (ChE) activities in order to compare the sensitivity of the two test systems. A wild type strain Oregon-R (Or-R) male flies of D. melanogaster were reared on a medium containing one concentration of each of VCR and parsley (4 ml/100 ml medium) in each single and combined treatment. Also the activity of ChE was estimated in some insects of the two generations: F1 females, F2 bar eye females (heterozygous) and F2 wild types males. The results indicate that both of parsley and vincristine did not cause significant increases of SLRL test in either the single or combined treatments. In contrast, estimation of ChE activities showed significant increase in all the broods within single and combination treatments, except female of the second generation of spermatid brood which treated with parsley and VCR at the same time. It is concluded that enzyme estimation is more sensitive than SLRL test for detection the mutagenic effect for parsley’s extract and vincristine.